Patent Application
20220146509
The present invention relates to a pregnancy diagnostic kit for diagnosing pregnancy more accurately by measuring human chorionic gonadotropin (hCG) as well as a variant thereof together in pregnancy diagnosis.
The immunochromatographic assay is a method capable of qualitatively and quantitatively analyzing a small amount of an analyte in a short period of time using an antigen-antibody reaction. Pregnancy diagnostic kits, which are widely used in homes and hospitals, use such an immunochromatographic assay. In the case of immunochromatography strips used in general pregnancy diagnostic kits, human chorionic gonadotropin (hCG), the amount of which increases in urine and serum during pregnancy, is used as a target substance to be detected. The antibody binding to hCG is immobilized on a membrane pad in a line form (test line), and another antibody is bound to a labeling material such as gold particles and the like and immobilized on a conjugate pad, and then the sample is developed. The developed sample passes through a conjugate pad and is developed by forming a complex of hCG-anti hCG antibody-gold particle in the sample, and the complex is captured by an anti-hCG antibody immobilized on the test line to form a ‘test line-immobilized antibody-anti hCG antibody-gold particle.’ This is called the “sandwich reaction”. When hCG is present at a concentration that can be determined as pregnancy in the sample, the test line appears red by gold particles, and it is determined as pregnant. In addition, the control line always captures the gold particle antibody regardless of the concentration of hCG in the sample, and since the red color appears by the gold particle, the normal termination of the test can be confirmed. The analysis strip showing the result can be detected by the naked eye or using a sensor.
Human chorionic gonadotropin (hCG) is a 36.5 kD glycoprotein, composed of two subunits, alpha (α) and beta (β), and it functions similarly to luteinizing hormone (LH), such as luteinizing of the ovary, inducing ovulation, promoting androgen secretion in interstitial cells of the testis, and the like. The hCG is produced and secreted from the placental trophoblast cells when a woman becomes pregnant and is present in the mother's blood, urine, and amniotic fluid. Other than the above, it is known to increase in trophoblastic diseases such as hydatidiform mole and choriocarcinoma and in various embryonic cell tumor patients, and it is also present in a very small amount in the blood and urine of normal men and non-pregnant women. The hCG is produced in the placenta and released through the blood into urine, and while passing through this step, various decomposition products and variants are generated, and there is a difference in the hCG forms shown in the placenta, blood, and urine. The a subunit has two N-nicked oligosaccharide side chains, and the β subunit distinguishes hCG from other glycol proteins in a unique form with 145 amino acid linkages on six legs. Depending on the change of this amino chain, the placenta exhibits the forms of large free α and non-nicked free β, and the forms of regular free a and nicked free β are added in the blood, and in particular, it is present in the form of hCG β core fragment (hCGβcf) in urine (Journal of the Korean Society of Emergency Medicine: 2011, Vol. 22, No. 5, 503-507).
In urine before 5 weeks of pregnancy, there are relatively few hCG β core fragments (hCGβcf), but in urine after 6 weeks of pregnancy, hCGβcf is present at a higher amount than normal hCG (intact hCG; I-hCG), and especially after 8 weeks, it is present at an amount 3 times or more than normal hCG. This high concentration of hCGβcf may interfere with the measurement of normal hCG and cause false negative errors in pregnancy diagnosis (J. Emergency Medicine 2013, 44:155).
Accordingly, as a result of continuously researching a method of manufacturing a pregnancy diagnostic kit capable of accurately determining whether to be pregnant without being affected by hCGβcf, which is present at a high concentration in urine, the present inventors completed the present invention by confirming that when the chromatography strip for diagnosing pregnancy having multiple test lines according to the present invention was used, it was possible to accurately determine whether to be pregnant even when a hCG variant was present at a high concentration.
Accordingly, an object of the present invention is to provide a chromatography strip for diagnosing pregnancy with high accuracy by excluding interference of hCG variants present at a high concentration in urine.
Another object of the present invention is to provide a pregnancy diagnostic kit including the chromatography strip.
Still another object of the present invention is to provide a method for diagnosing pregnancy using the chromatography strip or pregnancy diagnostic kit.
In one aspect for solving the above problems, the present invention provides a chromatography strip for diagnosing pregnancy including a conjugate pad and a detection pad.
In the chromatography strip for diagnosing pregnancy of the present invention, the conjugate pad includes a first anti-human chorionic gonadotropin (hCG) monoclonal antibody and a signal detection labeling material, the detection pad is provided with test line 1 and test line 2 that are isolated, the test line 1 includes a second anti-hCG monoclonal antibody, and the test line 2 includes human chorionic gonadotropin (hCG).
The “first anti-hCG monoclonal antibody” may be used interchangeably with the “first anti-hCG antibody” and refers to an antibody capable of recognizing the O-core fragment (hCGβcf) region of human chorionic gonadotropin. Therefore, the first anti-hCG monoclonal antibody of the present invention may specifically bind to a hCG variant including hCGβcf as well as normal hCG.
The “second anti-hCG monoclonal antibody” may be used interchangeably with the “second anti-hCG antibody”, and refers to an antibody capable of specifically binding by recognizing another region of hCG other than the hCGβcf region. Therefore, the second anti-hCG monoclonal antibody of the present invention may specifically bind to normal hCG only and may not bind to hCGβcf. In one preferred exemplary embodiment of the present invention, the other region of the hCG may be a part of the normal hCG β subunit.
In the immunochromatography strip for diagnosing pregnancy of the present invention, the first anti-hCG monoclonal antibody is linked to a signal detection labeling material before or during the development of a sample to form a first conjugate, and a complex in which hCG or a variant thereof is bound to the first conjugate is formed when hCG or a variant thereof is present in the sample. In addition, as the concentration of hCG or a variant thereof increases in the sample, the number of the formed complexes increases, and conversely, the number of bare conjugates which are not bound to hCG in the sample decreases.
A complex in which normal hCG is bound to the first conjugate is captured on test line 1 to indicate a signal, and a complex in which a variant is bound to the first conjugate is not captured on test line 1. In addition, a bare conjugate which is not bound to hCG or a variant thereof in the sample reacts with hCG immobilized at a certain amount on test line 2, thereby being captured on test line 2 to indicate a signal.
As used herein, the term “conjugate” refers to a conjugate formed by linking the labeling material and the ligand to each other, and the conjugate includes a first conjugate in which a labeling material for signal detection and a first anti-hCG antibody are linked to each other. The labeling material and the first anti-hCG antibody may be physically or chemically linked. That is, the labeling material and the first anti-hCG antibody may be linked by passive adsorption, and the labeling material may be modified to have a reactive group to be covalently bonded to the first anti-hCG antibody. However, it is not limited thereto, and the linkage of the labeling material and the first anti-hCG antibody may be performed using a method known to those skilled in the art. However, the labeling material and the first anti-hCG antibody may be present in the conjugate pad by being linked in the form of a conjugate before developing the sample on the chromatography strip for diagnosing pregnancy. Alternatively, they may be present in the conjugate pad in a separated state without being linked to each other, and as the sample is developed, they may be linked to each other to form a conjugate, or may exist in a solution phase and be added to the conjugate pad before use. In either case, during sample development, the labeling material and the first anti-hCG antibody may move to the detection pad in the state of a conjugate, and it is because they are not immobilized in the conjugate pad.
As used herein, the term “labeling material” refers to a material that generates a signal that may be detected by the naked eye or using a sensor. As the labeling material, gold colloid (gold particles), latex particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, luminescent materials, or magnetic particles may be used, but it is not limited thereto. In addition, the signal may be generated by itself due to the inherent characteristics of the labeling material such as light emission and the like, or may be generated by an external stimulus such as fluorescence and the like.
In the chromatography strip for diagnosing pregnancy of the present invention, the conjugate pad may further include a material that may be used as an internal control and a signal detection labeling material. The internal control material and the labeling material are linked to each other before or during sample development to form a second conjugate, and the second conjugate is captured by a ligand of the control line, thereby confirming the development of the sample.
As used herein, the term “control line” refers to a part that emits a constant signal regardless of the concentration of the sample or hCG in the sample. The control line may be formed using a method similar to the test line and the heterogeneous test line. However, while not binding to hCG, the control line may be formed by immobilizing a material that may be captured by specifically or non-specifically binding to a separate material bound to gold particles, as a second conjugate that moves along the detection pad by a mobile phase together with the sample. The material immobilized on the control line may be referred to as a ligand. As a result, it is formed by immobilizing a ligand capable of emitting a constant signal regardless of the presence or absence of chorionic gonadotropin in the sample. As the control line, anti-rabbit IgG, anti-chicken IgY, streptavidin, bovine serum albumin and the like may be used. The control line may be formed in two or more by varying the concentration of the ligand to be immobilized. The higher the concentration of the ligand immobilized on the control line, the more the control line may constantly emit a strong signal.
In the chromatography strip for diagnosing pregnancy of the present invention, the position of the test line and the position of the control line may be appropriately selected depending on the antigen-antibody reaction used and the like, but is not limited thereto.
Unlike the pregnancy diagnostic kit using a conventional analytical strip having a detection pad formed only with a control line that can confirm the development of a test line and a sample, the present invention is characterized in that by having two test lines consisting of test line 1 that detects only normal hCG and test line 2 that simultaneously detects a variant thereof, it may perform the determination of pregnancy more reliably.
In one exemplary embodiment of the present invention, when the first conjugate present in the conjugate pad moves to a liquid sample (such as urine of a woman of childbearing age, etc.) and a detection pad, it binds to the β-core fragment site of hCG in the sample to form a hCG-first anti-hCG antibody-gold particle complex. The complex is captured by a second anti-hCG antibody immobilized on test line 1 while being developed (sandwich immune reaction), and the first anti-hCG antibody-gold particle conjugate that is not bound to hCG in the sample is captured on test line 2 while being developed (competitive immune reaction). Since the amount of the first anti-hCG antibody-gold particle conjugate present in the conjugate pad is determined and used in a specific amount, as the concentration of hCG in the sample increases, it may show an inversely proportional relationship where the number of complexes, which are formed by the first anti-hCG antibody-gold particle conjugate present in the conjugate pad binding to hCG in the sample, increases, and conversely, the number of bare conjugates which are not bound to chorionic gonadotropin decreases. On the test line 1, the normal hCG β subunit may be measured by the sandwich immune method, and on the test line 2, the hCG and a derivative thereof may be simultaneously measured by the competitive immune method.
In one exemplary embodiment of the present invention, the signal intensity of the test line increases as the concentration of hCG in the sample increases, but when it increases above a certain concentration, it shows a phenomenon where the signal intensity rather decreases. The signal intensity from the test line 2, where the bare conjugate is captured, decreases as the concentration of hCG increases. In particular, it decreases under the influence of the hCGβcf concentration, which increases significantly after 6 weeks of pregnancy. As a result, it is possible to analyze hCG through signals from the labeling materials on the test line 1 and test line 2. More specifically, when the concentration of hCGβcf is low, it may not affect the sandwich immune test of test line 1, and thus, it is possible to accurately diagnose whether to be pregnant by measuring hCG with only test line 1. That is, when the test line 1 shows color development, it may be judged to be positive for pregnancy. However, when a large amount of hCGβcf is contained, it is possible to interfere with the immune test of the test line 1, and thus, the test line 1 and test line 2 may be combined to accurately diagnose pregnancy. That is, even when the test line 1 does not show color development, if the test line 2 is not visible or the intensity is below the control line, it is determined to be positive for pregnancy, and thus, it is possible to diagnose pregnancy more accurately.
The constitution of the chromatography strip for diagnosing pregnancy of the present invention will be described in more detail. The chromatography strip for diagnosing pregnancy may include a sample pad into which a sample to be confirmed whether a target substance is included; a conjugate pad having one end connected to the sample pad; a detection pad having one end connected to the other end of the conjugate pad; an absorbance pad having one end connected to the other end of the detection pad and providing a driving force for transferring the sample from the sample pad; and a solid support located under the chromatography strip for diagnosing pregnancy. The constitutional diagrams for the chromatography strip for diagnosing pregnancy of the present invention are shown in [
The solid support may be formed of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass, and plastic. By manufacturing by attaching a strip on the solid support, the durability of the strip may be increased, and handling and storage may be facilitated. In addition, it is possible to facilitate the mounting of an additional outer case. As the plastic material that may be used as the solid support, a polypropylene film, a polyester film, a polycarbonate film, an acrylic film, and the like may be used, but is not limited thereto.
In another aspect, as a kit in which the chromatography strip for diagnosing pregnancy is additionally fixed in a case, the present invention provides a diagnostic kit in which a guide and a strip support are provided in a lower case, and a sample input port is provided in an upper case; and a result confirmation window is provided at a position corresponding to multiple test lines and control lines.
The chromatography strip for diagnosing pregnancy may additionally be fixed in a case. The inside of the lower case may be provided with multiple guides and/or strip supports for positioning and fixing or compressing the chromatography strip for diagnosing pregnancy in an appropriate position. Optionally, the guide and the strip support may be provided in the upper case at positions corresponding to the guide and strip support provided in the lower case. That is, the guide and/or strip support may be formed in the lower case or both the upper case and the lower case, if necessary. In addition, the upper case may be provided with a result confirmation window for detecting a signal from the labeling material at a position corresponding to the sample input port, the test line, and the control line. The sample input port is formed at one end of the detection pad, that is, on the other end of the absorbance pad based on the test line, and at the same time, it may be formed in the form of a hole, a slit, and the like at a position sufficiently spaced apart with the test line such that the sample may be developed along a membrane. The result confirmation window may also include multiple test lines and control lines on the detection pad, and may be formed to a size large enough to be discernable from the outside by the naked eye or a sensor. As long as multiple test lines and control lines may be identified, the size and shape thereof may be formed without limitation.
The upper and lower cases may be manufactured using a conventional plastic material, for example, polycarbonate, acrylonitrile butadiene styrene (ABS), and the like may be used, but are not limited thereto. The upper and lower cases may be separately manufactured and provided with engaging grooves, engaging projections, and the like, and combined by conventional means, and in some cases, these may be integrally manufactured.
As described above, chromatography uses the principle of chromatography in which a mobile phase including an analyte moves along a medium. Therefore, for analysis using a chromatography strip for diagnosing pregnancy, a mobile phase is required to move a sample including an analyte along the strip. Accordingly, the analysis kit of the present invention may further include a buffer solution. The buffer solution not only acts as a mobile phase for moving the sample along the chromatography strip for diagnosing pregnancy, but may also serve as a solvent for dissolving the conjugate, and may also serve as a diluent for diluting the sample if necessary. In addition, for example, when performing a whole blood analysis, a component for dissolving (lysis) blood cell components such as red blood cells and the like may be further included. As the buffer solution, a conventional buffer solution such as a phosphate buffered solution (PBS) having a concentration of 10 mM to 1 M, a nonionic or amphoteric surfactant, a mixture thereof, or the like may be used without limitation, and depending on the type of desired reactions such as antigen-antibody reactions and the like, the composition and usage ratio of the buffer solution may be appropriately selected.
In another aspect, as a method for diagnosing pregnancy using the chromatography strip for diagnosing pregnancy, the present invention provides a method for diagnosing pregnancy performed by including injecting a sample into the conjugate pad or a pad positioned before to develop; and confirming the presence or absence of a signal of a labeling material from multiple test lines and control lines.
In performing a specific analysis method, it may proceed in the following order. First, a liquid sample may be injected into the conjugate pad or a pad positioned before. That is, the liquid sample may be injected directly into the conjugate pad, and the sample may be introduced into the strip, but may preferably be injected before the conjugate pad, for example, in the sample pad to be introduced. In addition, the sample may be uniformly mixed by adding a buffer solution such as PBS, and introduced into the strip in the same manner.
As described above, when the sample is loaded (introduced) onto the chromatography strip for diagnosing pregnancy, development begins, and then it may be confirmed whether the development of the sample is performed well through a control line. If the sample is well developed, it may confirm the presence or absence of the signal of the labeling material from several test lines and control lines. If it corresponds to 1) below, it may be diagnosed as non-pregnant, and if it corresponds to 2) to 4) below, it may be diagnosed as pregnant.
1) If the control line and test line 2 appear, this indicates that hCG or a variant thereof is not included in the sample, and it may be diagnosed as non-pregnant.
2) If test line 1, test line 2, and the control line all appear, this indicates that hCG or a variant thereof is included in the sample, and it may be diagnosed as pregnant.
3) If test line 1 and the control line appear, this indicates that hCG is included in the sample, and it may be diagnosed as pregnant.
4) If only the control line appears, this indicates that hCG or a variant thereof is included at a high concentration in the sample, and it may be diagnosed as pregnant.
As a sample for analysis of the present invention, it may include not only a general aqueous solution, but also all biological samples such as whole blood, blood cells, serum, plasma, bone marrow, sweat, urine, tears, saliva, skin, mucous membranes, hair, and the like isolated from mammals, preferably humans, and preferably, it may be urine.
In the present invention, a monoclonal antibody for detecting normal hCG and a variant thereof, which are present in the urine of a pregnant woman, are constituted to be paired with each other, and by overcoming the problem of false negatives due to the hCG variant, it is possible to perform pregnancy diagnosis more accurately and conveniently by the naked eye.
Furthermore, the concentration measurement range of hCG or a variant thereof is improved by using a chromatography strip for diagnosing pregnancy, which is composed of multiple test lines and control lines.
Hereinafter, the present invention will be described in more detail through exemplary embodiments. These exemplary embodiments are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these exemplary embodiments.
Manufacture of Immunochromatography Strips
1-1. Manufacture of Detection Pad Formed with Test Line. Heterogeneous Test Line, and Control Line
Three analysis lines were dispensed on a nitrocellulose membrane. The nitrocellulose film was laminated on a plastic card using a laminator. Afterwards, as a second anti-human chorionic gonadotropin antibody of test line 1, a monoclonal anti-human chorionic gonadotropin antibody (monoclonal anti-hCG 5014, Medix, Finland) was used, and human chorionic gonadotropin (hCG) was used for test line 2, and an anti-mouse immunoglobulin antibody from goat (anti-mouse antibody from goat, Arista, USA) was used as a ligand for the control line. Each of these was dispensed using an automatic dispenser and then dried at 25° C. to 30° C. for 2 days (48 hours).
1-2. Manufacture of Conjugate Pad
A conjugate pad was pre-treated by soaking a pad sufficiently with Tris buffer (10 mM, pH 8.5) containing 0.5% polyvinyl alcohol (PVA) and thoroughly drying in a dryer.
A first conjugate solution, in which colloidal gold particles with a diameter of about 40 nm were combined with a complex clone anti-human chorionic gonadotropin antibody (monoclonal anti-hCG 5006, Medix, Finland) as a first anti-chorionic gonadotropin antibody, was prepared, and a second conjugate solution, in which colloidal gold particles with a diameter of about 40 nm were combined with mouse immunoglobulin (mouse IgG, Arista. USA), was prepared. The first conjugate solution and the second conjugate solution were dispensed on a pre-treated conjugate pad, and after drying completely, it was prepared by cutting it to an appropriate size.
1-3. Manufacture of Sample Pad
A sample pad was sufficiently soaked in a 0.08 M borate buffer solution containing 1% Triton X-100, 0.5% NaN3, and 0.1% BSA, and after drying completely in a dryer, it was prepared by cutting it into an appropriate size.
1-4. Manufacture of Absorbance Pad
The absorbance pad was used as it was without any treatment in a state where moisture was completely dried in a dryer, and it was prepared by cutting it into an appropriate size.
1-5. Manufacture of Immunochromatography Strip
The detection pad, the conjugate pad, the sample pad, and the absorbance pad prepared through each of the above processes were assembled as shown in
That is, the sample pad was attached so as to overlap with one end of the conjugate pad, one end of the detection pad was attached so as to overlap with the other end of the conjugate pad, and the other end of the detection pad and one end of the absorbance pad were attached to overlap each other. It was cut to about 4±2.0 mm using a cutter to prepare a strip as shown in
In
1: Sample pad; 16±4×4±2 mm
2: Conjugate pad of first anti-hCG antibody and gold particles;
6±1.0×4±2 mm
3: Nitrocellulose detection pad; 25±5×4 f 2 mm
4: Plastic solid support
5: Absorbance pad; 18 4×4 2 mm
6: Test line 1 with immobilized second anti-hCG antibody
7: Control line with immobilized anti-mouse immunoglobulin
8: Test line 2 with immobilized hCG
1-6. Assembly of Case
After inserting the manufactured immunochromatography strip for diagnosing pregnancy into the fixed position of a strip in a plastic lower case, the upper case with a sample input port and a result confirmation window was inserted, and then assembled by pressing with a press.
Evaluation of Chromatography Strip for Diagnosing Pregnancy Using hCG Standard Solutions
Human hCG standard materials were spiked in non-pregnant urine to prepare standard samples of chorionic gonadotropin and subjected to quantitative and qualitative tests. The prepared standard sample solutions were dispensed by 100 μL into the sample input port of the case and then developed for 5 minutes. The sample solutions were prepared to have normal human chorionic gonadotropin concentrations at 0, 12.5, 25, 200, 5,000, 50,000, 500,000, 1,000,000, and 2,000,000 mIU/mL, respectively.
As shown in [Table 1] and [
Evaluation of chromatography strip for diagnosing pregnancy using sample solutions with hCG and hCGcf
Next, in order to confirm the accuracy of the analysis method of the present invention when pregnancy was diagnosed with urine including a β-core fragment, sample solutions were prepared that were spiked with a β-core fragment and a normal hCG standard material in non-pregnant urine, and these were developed for 5 minutes after dispensing by 100 μL. The sample solutions were prepared as shown in [Table 2] below.
As shown in [Table 2] and [
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0172576 | Dec 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/008465 | 7/10/2019 | WO | 00 |
